Title: Transendothelial transport mediated modulations to the High Density Lipoprotein Summary Despite the vast amount of literature that indicates low plasma levels of HDL-cholesterol (HDL-C) is a risk factor for cardiovascular disease (CVD), the last decade has seen a paradigm shift in the concept that it may not be HDL-C levels per se but the functionality of HDL that is a determining factor in CVD. The shift in focus is in light of several studies that show that neither pharmacological nor genetic intervention to increase HDL-C levels, lower the risk for CVD. Thus, there is a pressing need to fill the gap in knowledge regarding the role of HDL in CVD from a mechanistic perspective and understand structure-function relationships in HDL. In the current proposal, we hypothesize that apolipoprotein (apo) AI and apoE3, two critical apos on HDL, undergo structural alterations and post translational modifications (PTM) and that HDL undergoes particle modulation as a consequence of transendothelial transport from plasma to the arterial intima with significant functional penalties. We will test this hypothesis by interrogating structure-function changes to the HDL with two specific aims: (1) Identify structural and proteomic alterations to the HDL associated with transendothelial transport across aortic endothelial cells, and, (2) Determine changes in HDL function following transendothelial transport. We will carry out spectroscopic analysis of reconstituted HDL (rHDL) bearing spatially sensitive fluorescent probes at flexible locations on apoAI or apoE3 to obtain insight into conformational alterations following transcytosis. Based on previous findings about oxidatively modified apoAI in atherosclerotic plaques, we postulate that the apos are susceptible to oxidative modification during transcytosis. To address this, we will determine PTM in transcytosed HDL, specifically on apoAI, apoE3 and selected proteins involved in lipoprotein metabolism by mass spectrometry. We will also perform lipoproteomic analysis to identify changes to the protein and lipid composition of transcytosed HDL. In an independent but complementary approach, we will examine changes in two major functional effects of transcytosed HDL: its ability to promote cholesterol efflux from macrophages, and, its antioxidant activity. Completion of these studies will significantly advance our understanding of the relationship between structure/composition and the atheroprotective effect of HDL at the vascular wall. The expected outcome of the proposed studies is to have a deeper understanding of modulations in HDL that render it dysfunctional. Establishing this relationship will aid in development of HDL- based therapies and identification of biomarkers of CVD risk. The research enhancement objectives of the PI are to: (i) develop expertise in HDL and endothelial cell biology, (ii) increase competitiveness to apply for major external funding by publishing research findings, (iii) strengthen existing and develop new collaborations, submit joint publications and lay the groundwork for developing proposals, and, (iv) establish mentoring, networking, and leadership skills, and offer research training opportunities and mentorship for students from diverse backgrounds in biomedical research.